Summary of Work: The goal of this project is to identify the genes that interact with PAX3 to cause hearing impairment in individuals with Waardenburg syndrome (WS). Information obtained from studying PAX3 expression in a mouse model (Splotch) will be used to identify candidate genes which will then be analyzed in the DNA samples from human subjects with WS. In addition, individuals with WS who are hearing impaired are being screened for mutations in known DFN loci. In the past year a RACE library from 11.5 day mouse embryo tissue was constructed and 5' and 3' RACE (rapid amplification of cDNA ends) clones generated for both Pax3 and Pax7 (a gene very similar in structure and thought to form heterodimers with Pax3). Five different alternate transcript forms of Pax3 have been identified, all differing at the 3' end. Since the genomic structure of murine Pax3 has not been elucidated, we have identified 3 BAC clones that contain portions of the PAX3 cDNA): two span exons 5 to 8; and one contains exon 8 but not 5. The intron sequences immediately flanking exons 5 through 8 have been determined and sequences 3' to exon 8 are being established. Full length clones are being generated to express the Pax3, and eventually Pax7, protein isoforms and conduct DNA binding experiments. Probes designed to distinguish the 5 transcripts in ribonuclease protections assays (RPAs) are being constructed. A colony of Splotch and Splotch-delayed mice (mutant Pax3 alleles) has been established and embryos are being collected at relevant time points in inner ear development. These embryos will be used in: 1) differential display experiments to identify genes up- or down-regulated in response to a Pax3 mutation; 2) in situ and RPA experiments to establish the temporal and spatial patterns of expression of Pax3 isoforms and candidate genes for Pax3 regulation. Mutations in the GJB2 gene (= DNFA3 and DFNB1) have been identified in 3 unrelated WS individuals, suggesting that the hearing impairment in these three families may be the result of digenic inheritance.